The present invention relates to a gene transfer system comprising a ligand oligopeptide for growth factor receptor, an endosome-releasing oligopeptide and a polycationic polypeptide to form a complex with exogenous gene either in the form of free DNA of a recombinant eukaryotic expression vector or in the form of a recombinant viral vector. Via receptor mediated endocytosis, exogenous DNA was targetably transduced into the tumor cells for the purpose of tumor gene therapy.
Gene therapy is to transduce the exogenous DNA into certain type(s) of human cells for the treatment of human diseases. For the therapeutic purpose, it is of prime importance to generate a gene transfer system with safety and high efficiency and targetability. So far as it is concerned. there are two types of systems currently used to transfer exogenous genes into human cells. The first type is the viral vector system and the second is the non-viral vector techniques. However, no effective systems are available. Referring to the non-viral system, it was reported in recent years that exogenous DNA could be transduced into cells by receptor-mediated gene transfer techniques. The exogenous DNA can form a complex with a ligand associated polypeptide. After the binding of ligand and cell receptor, the DNA/polypeptide can be endocytosed thereby transferring the exogenous gene into cells enriched with the relevant receptors. Wu G. Y. et al (J. Biol. Chem. 263:14621, 1988) described that asiologlycoprotein conjugated polylysine can mediate uptake of exogenous gene by hepatocytes. Transferrin was also used as a ligand to transduce exogenous genes into cells by binding with cell surface transferrin receptor and subsequent endocytosis (Birnstiel M. L. et al, PNAS, USA. 87:3410-3414, 1990).
However, the gene transduction by these receptor-mediated gene transfer system was limited to non-tumorous cells, such as hepatocytes, but not described to target malignant cells. One of the major limitations of these vectors or other non-viral systems is the enzymatic degradation of the endocytosed DNA due to the fusion of lysosome and endosome vesicles, thereby reducing the transduction efficiency and expression of exogenous genes. However, it is well known that some envelope domains of virus, such as adenovirus or influenza virus, can cause endosomolysis and release the endocytosed virus, to protect from the lysosomal fusion and degradation. It was reported that defective or chemically inactivated adenovirus can be used to disrupt the endosome so as to increase the transfer efficiency of the receptor-mediated gene transfer system (Birnstiel M. L., PNAS, USA. 89:6094-6098, 1992).
The present invention is a novel targeting gene transfer system for tumor gene therapy. The present system comprises a ligand oligopeptide for receptor recognition (LOP), a polycationic polypeptide(PCP) for DNA binding and an endosome-releasing oligopeptide (EROP) for endosome disrupting. This system includes the 3-element composite polypeptide vector LOP/PCP/EROP which can bind DNA to form a 4-element complex gene transfer system and 2-element composite polypeptide vector LOP/PCP which can bind DNA to form a 3-element complex gene transfer system. The LOP includes E5(14 amino acid) for IGF-I and IGF-II receptor, GE7(16 amino acid) for EGF receptor and GV1(32 amino acid), GV2(36 amino acid) for VEGF receptor recognition. The PCP includes protamine, polylysine and histone. The EROP is a synthetic 20 amino acid oligopeptide homologous to haemagglutinin domain of Influenza viral envelope (HA20). To take advantage of the recognition capability of LOP for receptor overexpressed on surface membrane of cancer cells, therapeutic genes are to be transduced into cells via endocytosis and endosomolysis for tumor gene therapy.
It is an object of the present invention to provide a new gene transfer system that can targetably transduce exogenous genes into certain type of cancer cells in vitro and in vivo with high efficiency. This system consists of following elements:
i. A receptor-specific ligand oligopeptide(LOP) that is designed to recognize the cells with relevant expressed receptor.
ii. A polycationic polypeptide (PCP) for forming a complex with DNA from plasmid containing exogenous gene. The PCP is polylysine, protamine or histone.
iii. An endosome-releasing oligopeptide (EROP) for the release of exogenous DNA from endosome after endocytosis.
iv. The polypeptide comprising of the above elements to form a complex with DNA from recombinant eukaryotic expression vector containing exogenous gene as a novel gene transfer system (GDS).
The other object of the present invention is to provide the system that comprises composite polypeptide vector:
i. LOP and EROP are together linked with PCP to form LOP-PCP-EROP 3-element composite polypeptide vector
ii. LOP and EROP are independently linked with PCP to form LOP-PCP and EROP-PCP 3-element composite polypeptide vector.
The other object of the present invention is to design and prepare the LOP E5, GE7, GV1 and GV2:
i. E5 is to recognize and bind specifically to IGF-I and IGF-II receptors that are highly expressed in human hepatic or breast cancer.
ii. GE7 is to recognize and bind specifically to EGF receptor or indirectly to c-erbB2, that are highly expressed in human hepatic, breast, gastric, esophageal, lung cancer and brain glioblastoma.
iii. GV1 and GV2 are to recognize and bind to vascular endothelial growth factor receptor(VEGF R), which are highly expressed in angiogenetic vessels in most solid malignant tumors.
The other object of the present invention is to use protamine, polylysine and histone as a backbone of the GDS to form a complex with DNA.
The other object of the present invention is to use the composite polypeptide LOP-PCP and EROP-PCP to bind DNA and form a LOP-PCP/EROP-PCP/DNA complex. DNA of exogenous genes include:
1. Antisense sequence of protooncogenes rasH, rasK, rasN, c-myc, bcl-2 and growth factor receptor. The antisense sequence is either in a form of double stranded DNA or in a form of oligoribonucleotides or oligodeoxyribonucleotides.
2. Cancer suppressor gene p53 and Rb.
3. Suicide gene HSV-TK and CD.
4. Apoptosis-inducing gene p15, p16 and p21WAFxe2x88x921.
5. Cytokine gene GM-CSF, Interferon xcex1 and xcex3, Interleukin 2,3,4,12 and 15.
The other object of the present invention is to use the haemagglutinin domain oligopeptide HA20 as an element in GDS to break the endosome and prevent lysosomal degradation of transduced gene after endocytosis.
The HA20 is to be used either as a part of the covalently bound composite polypeptide, LOP-PCP/HA20-PCP to form a complex with DNA or as a free element used in conjunction with LOP-PCP/DNA complex.
The another object of the present invention is to utilize the GDS to transduce genes into cells either in vitro or in vivo for human tumor gene therapy.
The present invention principally relates to 4 parts: providing ligand oligopeptides (LOP) for specific recognition and binding to cell growth factor receptors; providing a 2-element composite polypeptide vector LOP-PCP to form a 3-element gene transfer system composed of a complex of DNA from exogenous gene and LOP-PCP; providing a 3-element composite polypeptide vector LOP-PCP-EROP to form a 4-element gene transfer system composed of a complex of DNA from exogenous gene and LOP-PCP-EROP or LOP-PCP plus EROP-PCP; providing methods and techniques to transduce gene(s) into cells both in vitro and in vivo for tumor gene therapy.
1. The first part of the present invention is to provide the LOP E5, GE7, GV1 and GV2 for specific recognition and binding to insulin growth factor I and II receptor(IGF-I R and IGF-II R), epidermal growth factor receptor(EGF R) and vascular endothelial growth factor receptor(VEGF R) respectively.
It has been demonstrated that IGF-I R and IGF-II R are over-expressed in human hepatic cancer and other malignancies. EGF R is highly expressed in human hepatic, mammary, ovarian, gastric, cervix cancer, glioblastoma, lung adenocarcinoma, nasopharyngeal cancer etc. VEGF R is over-expressed in vascular endothelial cells of tumor blood vessels and some cancer cells. Based on the mechanism of growth factor ligand and its receptor recognition and binding, a series of LOPs for above-mentioned receptors are designed and synthesized by using Peptide Synthesizer 430(ABI). The procedures of oligopeptide synthesis are according to the manufacturer""s manual. The LOP products are purified by high pressure liquid chromatography and lyophilized. The product quality has been confirmed by amino acid analysis.
Synthesis of 4-element complex gene transfer system was performed and then subjected to biological and screening analysis. First of all, LOP and PCP (polylysine, protamine or histone) were independently reacted with SPDP(N-esuccinimidyl-3-2-pyridyl-dithiopropionate) to produce LOP-3-(2-pyridyldithio)-propionate (LOP-PDP) and PCP-PDP. PCP-PDP was then converted into PCP-(SH)2 and form LOP-PCP composite polypeptide vector with LOP-PDP by disulfide covalent bond. FIGS. 1-3 indicated the Sephadex G50 chromatography purification of E5-polylysine, GE7-polylysine and GV2-protamine conjugates respectively. The first peak was collected as the purified product. FIGS. 4 and 5 demonstrated the results of amino acid composition analysis of the hydrolyzate of E5-oligopeptide and GE7-polylysine. Using the same method, EROP(HA20)-PCP was prepared. A 4-element DNA/polypeptide gene transfer system composed of LOP-PCP/EROP-PCP/DNA was prepared based on the electrostatic binding between negative charge of DNA and the positive charge of PCP in the 3-element composite polypeptide vector LOP/PCP/EROP. Using xcex2-galactosidase(pSV-xcex2-gal) as an exogenous gene, the transition of LOP-PCP/HA20-PCP/xcex2-gal into human hepatoma cells was performed in vitro and the transduction efficiency was examined by X-gal(5-bromo-4-chloro-3-indolyl-xcex2-D-galactoside) cytochemistry analysis. E5, GE7, GV1 and GV2 were proved to be effective LOPs for receptors IGF-I R and IGF-II R, EGF R and VEGF R respectively. These receptors were either highly expressed in cancer cells or in tumor vascular endothelial cells. FIGS. 6-8 were the results of agarose electrophoresis of E5, GE7, GV1 and GV2 4-element gene transfer system respectively, indicating DNA migration retarded by polypeptide as a stable complex. FIGS. 9-12 illustrated the results of E5 4-element gene transfer system to transduce xcex2-gal gene into human hepatoma cell line SMMC-7721 revealed by X-gal(blue granules in cells). In contrary, E5 system can not transduce xcex2-gal gene into normal hepatocyte cell line L02 or normal primary culture hepatocyte R02, similar to PBS control(no blue granules in cells). GE7 4-element system can transduce xcex2-gal gene into human hepatoma BEL-7402 (FIG. 13) and GV2 system transduced xcex2-gal gene into tumor vascular endothelial cells (FIG. 14).
Based on the biological screening assay, the LOPs in the present invention are E5(amino acid sequence, EPFRS PDLAL ETYG), (SEQ ID NO.1) GE7(NPVVG YIGER PQYRD L), (SEQ ID NO.2) GV1(CHPIE TLVDI FQEYP DEIEY IFKPS PVPLM RP), (SEQ ID NO.3) GV2(PVPTE ESNIT MQIMR IKPHQ GQHIG EMSFL QHNKC E), (SEQ ID NO.4).
Any oligopeptides that possess similar immuogenic epitopes with E5, GE7, GV1 and GV2 oligopeptides to react with antibodies against the above mentioned oligopeptides and that can bind with IGF-I R, IGF-II R, EGF R and VEGF R is adapted to the present invention. Any oligopeptides that some of the amino acids of the above mentioned oligopeptides were replaced by similar amino acids in corresponding site but not affecting their ability to bind the receptor is also adapted to the present invention.
The LOP E5, GE7, GV1 and GV2 mentioned above can be in the form of nucleotide sequence, used to construct a recombinant expression vector for the purpose of construction of a gene transfer system LOP/PCP/HA20 or to construct a hybrid gene with toxin or cytokine for therapeutic use.
The present invention is also to use LOPs mentioned above to recognize and bind to receptor(s) that are highly expressed in hemopoietic cells, macrophage, lymphocytes, hepatic, renal, endothelial, neural and cardiac muscle cells and to use these LOP to construct LOP-PCP/HA20-PCP for transducing exogenous gene into the above mentioned relevant cells for gene therapy of cancer or non-cancer disease.
2. The second part of the present invention is to provide a 2-element composite polypeptide vector LTOP-PCP to form a 3-element complex gene transfer system as a complex of LOP-PCP and DNA from exogenous gene. The LOP includes E5, GE7, GV1 and GV2; PCP includes protamine, polylysine or histone. DNA exogenous genes include cancer suppressor gene, suicide gene, apoptosis-inducing gene, cytoktine gene and antisense sequences of protooncogens constructed in an eukaryotic expression vector driven by SV40 or CMV promotors, or constructed in recombinant retrovirus or adenovirus. Antisense sequences include those derived from protooncogens(rasH, rasK, rasN, c-myc, bcl-2), growth factors and their receptor genes. Antisense sequence relate either to DNA construct of a complete or part of the cDNA, or to single-stranded oligonucleotides. Cancer suppressor genes include p53 and Rb. Suicide genes include HSV-TK(Herpes simplex thymidine kinase), CD(cytosine deaminase)genes. Apoptosis-inducing genes include p15, p16, and p21WAFxe2x88x921. Cytokine genes include GM-CSF(Granulocyte Macrophage Colony Stimulating Factor), TNF xcex1 (Tumor necrosis factor xcex1), IFN(Interferon) xcex1, xcex3, IL(Interleukin) 2,3,4,12,15 gene. The preparation of DNA/LOP-PCP is similar to that described in the previous section(Part 1). LOP is covalently linked to PCP (protamine, polylysine or histone) to form LOP-PCP. DNA and LOP-PCP are electrostatically bound to form a LOP-PCP/DNA complex as a 3-element complex gene transfer system.
3. The third part of the present invention is to provide a 3-element composite polypeptide vector of LOP-PCP-EROP to form a 4element complex gene transfer system as a complex of LOP-PCP-EROP and DNA from exogenous gene. The present invention also provides a 3-element composite vector as LOP-PCP polypeptide plus EROP-PCP polypeptide to form a 4-element complex with DNA as LOP-PCP/EROP-PCP/DNA. The presence of EROP in the vector increases the transduction and expression efficiency of exogenous gene. EROP in the present invention is to take a synthetic oligopeptide HA20 homologous to the haemagglutinin domain of Influenza virus. The synthesis of HA20 refers to literature (Midoux P. Nucleic Acid Res., 21:871,1993). HA20 is a 20 amino acid oligopeptide with its sequence as GLFEA IAEFI EGGWE ELIEG, (SEQ ID NO.5).
The first step of preparation of the 4-element gene transfer system is to covalently link LOP with PCP (protamine, polylysine or histone), by using coupling reagent SPDP. Then, HA20 is covalently linked the LOP-PCP to form LOP-PCP-HA20 3-element composite polypeptide vector. The LOP-PCP-HA20 is mixed and reacted with DNA from exogenous gene to form a complex of LOP-PCP-HA20/DNA by electrostatic binding. By similar method, HA20 is linked with PCP(polylysine, protamine or histone) to form HA20-PCP. LOP-PCP and HA20-PCP are mixed according to certain molar ratio and react with DNA from exogenous gene to form a LOP-PCP/HA20-PCP/DNA complex by elecrostatic binding. FIGS. 6,7,8 demonstrated results of agarose gel electrophoresis of the E5, GE7, GV1 and GV2 4-element LOP-PCP/HA20-PCP/DNA gene transfer systems, respectively, indicating the migration retardation of the stable complex. HA20-PCP can be mixed with LOP-PCP and reacted with DNA from exogenous gene to form 4-element complex gene transfer system LOP-PCP/HA20-PCP/DNA. In addition, HA20 oligopeptide can be used by mixed with LOP-PCP/DNA. All these preparation can increase the gene transfer efficiency.
The 4-element gene transfer system is also adapted for using two or more than two different types of LOP in LOP-PCP/HA20-PCP system to form complex with DNA from exogenous gene or recombinant virus. In addition, it is also adapted for using 4-element system of same type of LOP to form a complex with DNA or recombinant virus derived from two or more than two different types of exogenous genes for the purpose of cancer gene therapy.
The 3-element complex gene transfer system is further to use the 2-element composite polypeptide vector LOP-PCP to form a conjugate with recombinant virus for gene therapy. The recombinant virus includes genetic engineered adenovirus or retrovirus containing exogenous genes. Recombinant viruses can be either in a conjugated or non-conjugated form in the 3-element gene transfer system. The genes in recombinant viruses include suicide gene HSV-TK, CD; apoptosis-inducing gene p15, p16, p21WAFxe2x88x921; cancer suppressor gene p53, RB; cytokine gene GM-CSF, TNF xcex1, INF xcex1, xcex3, IL 2, 3, 4, 12, 15; antisense sequence of protooncogene rasH, rasK, rasN, c-myc, bcl-2; antisense sequence of growth factor and receptor gene, IGF-I, IGF-II, EGF, IGF-I R, IGF-II R, EGF R. The present system can also use defective virus that does not contain any exogenous gene. The present system can also apply in antisense oligoribonucleotides or oligodeoxyribonucleotides.
The above mentioned 3-element polypeptide LOP/PCP/EROP can be produced by genetic engineering techniques by constructing a recombinant DNA expression plasmid that encodes the amino acid sequence of HA20, PCP and EROP, thus facilitating the industrial production of vector system of well-controlled quality.
4. The forth part of the present invention is to apply the 3-element LOP/PCP/EROP composite polypeptide vector and its capability to form DNA/polypeptide gene transfer system in gene transfer both in vitro and in vivo for tumor gene therapy.
In vitro Experiment
1) In vitro gene transfer E5-polylysine/HA20-polylysine/xcex2-gal 4-element complex gene transfer system has been applied to transduce xcex2-gal gene into human hepatoma cell line SMMC-7721, normal hepatocyte line L02 and normal primary culture hepatocyte R02. Results indicated that xcex2-gal gene was transferred into hepatoma cells mediated by the 4-element gene transfer system with high efficacy and targetability (FIGS. 9-12). As control, xcex2-gal DNA alone. E5-polylysine/xcex2-gal 3-element complex, HA20-polylysine/xcex2-gal 3-element complex, polylysine/xcex2-gal and PBS were also used for transfection IGF-I R and IGF-II R positive SMMC-7721 cells. The transduction efficacy and targetability were evaluated by X-gal stain. Results were illustrated in Table 1-3.
The transduction efficiency of the complex derived from different elements was shown in table 1. The 4-element system can transduce xcex2-gal gene into hepatoma SMMC-7721, but not into normal human hepatocyte line L02 and primary culture hepatocyte R02 (Table 2). Table 3 illustrated that xcex2-gal into SMMC-7721 cells can be detected at 48 hr after transduction and reached the peak at 72 hr.
GE7-polylysine/HA20-polylysine/xcex2-gal or GE7-protamine/HA20-protamine/xcex2-gal 4-element gene transfer system was used to transfer xcex2-gal gene into different types of human cancer cell lines in vitro, including human hepatoma SMMC-7721 and BEL-7402, glioblastoma U87, mammary cancer Bcap-37, ovarian cancer 3A0 and A0, pulmonary adenocarcinoma SPC-A1. Normal hepatocyte L02 and mouse NIH/3T3 were used as control. As illustrated in FIGS. 13 and 15-22, xcex2-gal gene was transduced by GE7 4-element gene transfer system into EGF R positive cell lines: BEL-7402, SMMC-7721, U87, Bcap-37, 3A0,A0, SPC-A1 revealed b X-gal stain. GE7 4-element system failed to transduce xcex2-gal gene into EGF R negative cell line L02 or NIH/3T3, as no blue granules were observed by X-gal. xcex2-gal gene can not be efficiently transduced into EGF R positive BEL-7402 by 3-element complex HA20-polylysine/xcex2-gal, 2-element complex polylysine/xcex2-gal or xcex2-gal naked DNA(FIGS. 23-25).
The transduction rate of xcex2-gal into BEL-7402 cells by GE7 3-element system(GE7-polylysine/xcex2-gal) was less than 10% (FIG. 26). Taking naked xcex2-gal DNA, 3-element complex GE7-polylysine/xcex2-gal, HA20-polylysine/xcex2-gal, 2-element complex polylysine/xcex2-gal and PBS as control, the efficiency of the gene transfer system was examined on EGF R positive cell line BEL-7402 with X-gal histochemical stain. The result was listed in Table 4 and 5. The transduction efficiency of GE7 4-element complex to different cell line and the transduction efficiency of the complex composed of different elements to hepatoma cell line BEL-7402 was demonstrated in Table 4. The transduction efficiency of GE7 4-element system in transferring xcex2-gal gene into hepatoma versus normal hepatocytes and mouse NIH/3T3 was illustrated in Table 5. In Table 6, it was demonstrated that xcex2-gal gene expression initiated at 24 hr after transduction and reaches its peak at 168 hr in BEL-7402 mediated by GE7 4-element system.
1) In vitro transduction efficacy of apoptosis-inducing genes, p21WAFxe2x88x921, p16 and p15 to inhibit growth of human hepatoma cells mediated by E5 4-element gene transfer system.
E5-polylysine/HA20-polylysine/p21WAFxe2x88x921system was added into medium of cultured human hepatoma SMMC-7721 cells for 72 hr. After fixed with acetone, cells were stained with DAPI to examine the cell nuclear structure. As shown in FIG. 27, chromatin condensation, nuclear pyknosis and fragmentation as characteristic features for apoptosis, were observed. Using in situ terminal end-labeling techniques by Boehringer Mannheim Kit, apoptosis was obviously detected as shown in FIGS. 28 and 29. At 96 hr. after transfection, apoptosis was observed in 15% of cells mediated by p21WAFxe2x88x921E5 4-element system; negative results were observed in control without p21WAFxe2x88x921. Results of growth inhibitory effect on 7721 cells by E5 4-element system containing p21WAFxe2x88x921, p16 and p15 gene were demonstrated by the growth curve of cells cultured in 24 well-plate and counted at 2 day intervals(FIG. 30).
The GE7-polylysine/HA20-polylysine/p21WAFxe2x88x921 4-element gene transfer system was used to transduce human hepatoma BEL-7402 cells. 5 and 7 days after transfection, DNA was isolated from cells, both the suspended and adhered, and analyzed in 1.5% agarose gel electrophoresis. DNA ladder was observed in the transduced group, but not in the control, indicating that apoptosis was induced by p21WAFxe2x88x921 gene transduced into the hepatoma cells. Using the same system, cell counts were performed at day 1, 2,3, 4, 5, 6, 7, 8 after transfection. Results indicated that p21WAFxe2x88x921 can effectively inhibit the growth of hepatoma cells mediated by the GE7-polylysine/HA20-polylysine/p21WAFxe2x88x921 4-element system. The growth curve was demonstrated in FIG. 31, with PBS as control.
In vivo Experiment
1) In vivo gene transduction E5-polylysine/HA20-polylysine/xcex2-gal 4-element gene transfer system was used to transduce gene into human tumor in vivo. Human hepatoma SMMC-7721 was transplanted subcutaneously in nude mice. The above system was administrated by injection subcutaneously around the tumor. Animals were sacrificed at 12, 24, 48, 60,72 and 96 hr after treatment. Tumors were dissected and stained with X-gal. Results demonstrated that xcex2-gal expression was observed at 12 hr, reached its peak at 24 hr and decreased from 48 hr. Expression was still detected at 96 hr (FIG. 32).
GE7-polylysine/HA20-polylysine/xcex2-gal system was administrated in the same way into different human tumors subcutaneously implanted in nude mice. The human tumors included hepatoma SMMC-7721, BEL-7402, brain glioma U251, breast cancer Bcap-37, ovarian cancer 3A0, lung adenocarcinoma SPC-A1, colon cancer LOVO, gastric cancer SGC, lung small cell carcinoma H128 and cervical cancer xenograft. Animals were sacrificed at 4, 8, 12, 24 hr, day 2, 4, 7, 15, 20, 30 and 40. Tumors were dissected then and stained with X-gal. Results were illustrated in FIG. 33, indicating that xcex2-gal gene can be effectively transduced into all above human tumors except H128 which has been proved to be negative for EGF R. FIGS. 34 to 42 illustrated the results of histochemistry examination of xcex2-gal expression in these human cancer. Positive results were observed in all these human tumors (FIGS. 34-41) except H128 (FIG. 42).
GV2-protamine(or histone)/HA20-protamine/xcex2-gal 4-element gene transfer system was injected under microscope into portal vein of nude mice in which human hepatoma was intrahepatically transplanted. Animals were sacrificed at day 14 after treatment. Tumors were dissected and stained with X-gal. The frozen section were counterstained with Fast Nuclear Red and examined under microscope. xcex2-gal gene expression was observed in endothelial cells in capillaries and small blood vessels in tumor, particularly in regions close to necrosis lesion inside the tumor. xcex2-gal expression was also detected in some infiltrating cancer cells(FIG. 43). Low level of expression of xcex2-gal gene has been detected in endothelial cells of large blood vessels. No xcex2-gal expression was detectable in normal liver cells. GV2-protamine/HA20-protamine/xcex2-gal was also injected into hepatoma that was implanted subcutaneously in nude mice. Tumor was dissected from animals sacrificed two days later and stained with X-gal. Histochemical study of frozen section demonstrated that xcex2-gal was expressed in endothelial cells of capillaries and small blood vessels of tumor(FIG. 44), but not in liver cells.
2) Efficacy test of inhibition of tumor growth in vivo by transduction of p21WAFxe2x88x921.
Human hepatoma SMMC-7721 was subcutaneously implanted in nude mice. After tumor size reached about 0.5 cm, animals were randomly divided into different groups, 6 mice per each. Animals received treatment of injection of E5-polylysine/HA20-polylysine/p21WAFxe2x88x921 or E5-polylysine/HA20-polylysine around tumor twice per week, for two weeks. The dose per injection was equivalent the complex containing 20 xcexcg of plasmid p21WAFxe2x88x921 DNA. Animals were sacrificed 3 weeks after treatment. Tumors were dissected, weighed and their size was measured. Results were summarized in Table 7 and FIG. 45, indicating a significant inhibitory effect on growth of hepatoma by transduction of p21WAFxe2x88x921 in vivo, mediated by E5-plylysine/HA20-polylysine/p21WAFxe2x88x921 system as compared with the E5 3-element system in terms of the tumor volume and weight (Table 7).
As no inhibitory effect on tumor growth was observed in groups treated with E5-polylysine/p21WAFxe2x88x921, E5-polylysine/HA20-polylysine, HA20-polylysine/p21WAFxe2x88x921, polylysine/p21WAFxe2x88x921 and p21WAFxe2x88x921 DNA alone, it was demonstrated that E5-polylysine/HA20-polylysine/p21WAFxe2x88x921 4-element complex gene transfer system can effectively deliver exogenous gene into hepatoma cells in vivo and significantly inhibit the growth of hepatoma in vivo.
Using the same nude mice model with subcutaneously transplanted human hepatoma, GE7-polylysine/HA20-polylysine/p21WAFxe2x88x921 4-element complex system was injected subcutaneously at the site surrounding the tumor after it reached the size of 0.5 cm in diameter. The dose per mouse was the amount of complex containing 0.2 xcexcg p21WAFxe2x88x921 DNA, one injection only. Normal saline, GE7-polylysine/HA20-polylysine polypeptide and p21WAFxe2x88x921 DNA alone were injected as control. Animals were sacrificed two weeks after treatment. The weight and volume of the dissected tumors were measured. Results indicated the significant inhibitory effect of the p21WAFxe2x88x921 gene mediated by GE7 4-element gene transfer system on growth of human hepatoma in vivo(FIG. 46, Table 8 and 9). Table 8 and 9 demonstrated the inhibitory effect was significant based on both the data from measurement of tumor volume(Table 8) and weight(Table 9), thereby indicating that exogenous gene can be effectively target to transplanted human hepatoma and inhibit the growth of hepatoma cells in vivo.
According to the ability of the polypeptide vector system to bind the recombinant virus containing various types of exogenous genes, the present invention possesses the potential applicability to use multiple exogenous genes in gene therapy.
Based on the presented experimental data, the present invention possesses the capability to target DNA of exogenous gene(s) into tumor cells both in vitro and in vivo to inhibit the growth of tumor, thereby indicating a broad potential in cancer gene therapy.
The novelty of the present invention was as following:
1. It is the gene transfer system firstly described in utilizing ligand oligopeptide(LOP) constructed in a 2-element or 3-element composite polypeptide vector to target exogenous genes to cancer cells or tumor vascular endothelial cells in which certain types of receptors are over-expressed.
2. It is a first description to use protamine as a polycationic polypeptide(PCP) backbone to form a complex with DNA and to use the endosome releasing oligopeptide HA20 as a component of composite polypeptide vector system.
3. It is a first description to integrate the LOP, PCP and EROP to construct a composite polypeptide vector that has effectively targeted exogenous genes to cancer cells and significantly inhibited the growth of cancer cells both in vitro and in vivo.
The present invention provides a set of LOP including E5, GE7, GV1 and GV2 for constructing LOP/PCP 2-element composite polypeptide vector to form a 3-element complex with DNA of exogenous gene, and for constructing LOP/PCP/EROP 3-element composite polypeptide vector to form a 4-element complex with DNA of exogenous gene as a gene transfer system. The presence of EROP can effectively increase the transduction efficiency. The HA20, a homologue of Influenza hemagglutinin, can be used as an EROP either in its free form or in a conjugated form covalently linked to PCP.
The present invention provides a novel non-viral vector system to target DNA of exogenous gene to cancer cells both in vitro and in vivo and to kill cancer cells but not the adjacent and distant normal cells. Its high efficiency and targetability makes it possible to transduce exogenous gene to cancer cells for treatment of cancer.
The present invention can transduce DNA of the exogenous gene with a size from 10 to 104 nucleotides, thereby breaking through the size limitation of exogenous gene transduction by viral vectors, particularly for transferring large DNA sequences containing inducible regulatory sequence, multiple genes and genes of unusual size. The potential application of the present vector system to bind with recombinant virus is to further explore the area of application.
The present invention is to use one type of LOP-containing 3-element composite polypeptide vector to target two or more types of exogenous genes for multiple gene therapy.
The present invention is also to use two or more types of LOP 3-element composite polypeptide vectors to target one type of exogenous gene for further enhancing the efficiency of gene transduction into cancer cells.
In addition, the present invention is to construct a recombinant DNA expression vector, which containing the coding sequence of LOP/PCP/EROP. The polypeptide expressed in E. Coli, yeast or mammalian cells is feasible for large-scale industrial production.
Furthermore, the present invention is also adaptable to transduce exogenous genes to hemopoietic, lymphoid, liver, kidney, nerve cells with specific LOP, which can recognize the relevant receptors expressed in these cells for gene therapy of diseases including cancer, genetic, cardiovascular, neural, renal diseases and liver function failure.